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. 1996 Jun;62(6):1873–1879. doi: 10.1128/aem.62.6.1873-1879.1996

Cytochemical colocalization and quantitation of phenotypic and genotypic characteristics in individual bacterial cells.

A S Whiteley 1, A G O'Donnell 1, S J Macnaughton 1, M R Barer 1
PMCID: PMC167965  PMID: 8787385

Abstract

The widely accepted view that most bacterial species have yet to be cultivated in vitro has gained support from recent ribosomal DNA-based environmental studies. To enable elucidation of the phenotypes of organisms recognized solely by molecular genetic techniques, we developed and evaluated cytochemical methods which colocalize phenotypic properties with in situ rRNA probe hybridization signals. Application of these methods to artificial mixtures of Pseudomonas putida and Escherichia coli or Vibrio vulnificus showed that biochemical properties, such as the cytochrome oxidase reaction and specific substrate-enhanced tetrazolium salt reduction, can be assigned to cells identified by signals from determinative fluorescent rRNA probe binding. By doing the reactions directly on the stage of an inverted microscope and monitoring reaction product formation with a charge-coupled device video camera, it was possible to determine the kinetics of oxidizable substrate utilization in single cells. Analysis of digitized images permitted quantitative study of the relationship between rRNA signal strength and the rate of tetrazolium salt reduction. The approach used in this study opens up new opportunities to investigate the biochemistry, physiology, and behavior of both culturable and nonculturable bacteria in their natural environments.

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Selected References

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  1. Amann R. I., Krumholz L., Stahl D. A. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol. 1990 Feb;172(2):762–770. doi: 10.1128/jb.172.2.762-770.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barer M. R., Marsh P. J. Rapid cytochemical demonstration of cytochrome oxidase activity in pathogenic bacteria. J Clin Pathol. 1992 Jun;45(6):487–489. doi: 10.1136/jcp.45.6.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barer M. R. New possibilities for bacterial cytochemistry: light microscopical demonstration of beta-galactosidase in unfixed immobilized bacteria. Histochem J. 1991 Nov-Dec;23(11-12):529–533. doi: 10.1007/BF01041179. [DOI] [PubMed] [Google Scholar]
  4. DeLong E. F., Wickham G. S., Pace N. R. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science. 1989 Mar 10;243(4896):1360–1363. doi: 10.1126/science.2466341. [DOI] [PubMed] [Google Scholar]
  5. Fuhrman J. A., McCallum K., Davis A. A. Novel major archaebacterial group from marine plankton. Nature. 1992 Mar 12;356(6365):148–149. doi: 10.1038/356148a0. [DOI] [PubMed] [Google Scholar]
  6. Gribbon L. T., Barer M. R. Oxidative metabolism in nonculturable Helicobacter pylori and Vibrio vulnificus cells studied by substrate-enhanced tetrazolium reduction and digital image processing. Appl Environ Microbiol. 1995 Sep;61(9):3379–3384. doi: 10.1128/aem.61.9.3379-3384.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kemp P. F., Lee S., Laroche J. Estimating the growth rate of slowly growing marine bacteria from RNA content. Appl Environ Microbiol. 1993 Aug;59(8):2594–2601. doi: 10.1128/aem.59.8.2594-2601.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Nwoguh C. E., Harwood C. R., Barer M. R. Detection of induced beta-galactosidase activity in individual non-culturable cells of pathogenic bacteria by quantitative cytological assay. Mol Microbiol. 1995 Aug;17(3):545–554. doi: 10.1111/j.1365-2958.1995.mmi_17030545.x. [DOI] [PubMed] [Google Scholar]
  9. Poulsen L. K., Ballard G., Stahl D. A. Use of rRNA fluorescence in situ hybridization for measuring the activity of single cells in young and established biofilms. Appl Environ Microbiol. 1993 May;59(5):1354–1360. doi: 10.1128/aem.59.5.1354-1360.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Roslev P., King G. M. Application of a tetrazolium salt with a water-soluble formazan as an indicator of viability in respiring bacteria. Appl Environ Microbiol. 1993 Sep;59(9):2891–2896. doi: 10.1128/aem.59.9.2891-2896.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Schaule G., Flemming H. C., Ridgway H. F. Use of 5-cyano-2,3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water. Appl Environ Microbiol. 1993 Nov;59(11):3850–3857. doi: 10.1128/aem.59.11.3850-3857.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Schmidt T. M., DeLong E. F., Pace N. R. Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing. J Bacteriol. 1991 Jul;173(14):4371–4378. doi: 10.1128/jb.173.14.4371-4378.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Stackebrandt E., Liesack W., Goebel B. M. Bacterial diversity in a soil sample from a subtropical Australian environment as determined by 16S rDNA analysis. FASEB J. 1993 Jan;7(1):232–236. doi: 10.1096/fasebj.7.1.8422969. [DOI] [PubMed] [Google Scholar]
  14. Thom S. M., Horobin R. W., Seidler E., Barer M. R. Factors affecting the selection and use of tetrazolium salts as cytochemical indicators of microbial viability and activity. J Appl Bacteriol. 1993 Apr;74(4):433–443. doi: 10.1111/j.1365-2672.1993.tb05151.x. [DOI] [PubMed] [Google Scholar]
  15. Walker D. R., Nwoguh C. E., Barer M. R. A microchamber system for the rapid cytochemical demonstration of beta-galactosidase and other properties in pathogenic microbes. Lett Appl Microbiol. 1994 Feb;18(2):102–104. doi: 10.1111/j.1472-765x.1994.tb00816.x. [DOI] [PubMed] [Google Scholar]

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